The simultaneous identification of multiple novel disease loci in genome wide association studies (GWAS) has highlighted a need for efficient model systems both to define the causal gene(s) at each locus, and to explore disease mechanisms 2, 3. Many GWAS have explained only a small proportion of the heritability for the traits of interest, and for some key disease phenotypes no loci have been identified2-4. There is a clear need for rapid in vivo approaches to vertebrate gene function, but also an imperative to begin to identify intermediate phenotypes more proximate in the disease causal pathways11-13. We have taken an integrative approach combining quantitative intermediate phenotypes for major cardiovascular traits, specifically;left ventricular LV hypertrophy, arterial calcification scores and key metabolites, from the Framingham Heart Study (FHS) with cognate validated functional assays in the intact zebrafish14-20. We will explore the use of high throughput zebrafish genetics for gene identification at GWAS loci, for prioritization of loci of borderline significance, and for investigation of gene-gene and gene-drug as well phenotypic interactions in the following Specific Aims: Aim 1 Identify the causal genes at GWAS loci for cardiovascular traits in FHS using in vivo modeling in the zebrafish: All genes at each GWAS locus (within 500kb of the original genetic signal) for three core phenotypes in the FHS will be tested using a range of multiple cognate assays in the zebrafish and loss of function and over-expression alleles for each gene. The human phenotypes and their zebrafish counterparts are;LV mass: in vivo myocardial mass, myocardial hypertrophy reporters and LVH expression profiles;Arterial calcification indices: in vivo vascular structure and physiology, aortic LDL transport and NO production, Wnt and Id reporters of vascular calcification pathways;and Metabolomics: direct comparisons of human and zebrafish metabolites. These experiments will define the major effect gene(s) at many of the loci for each phenotype, the direction of biologic effect and potential relations across shared intermediate phenotypes 11,12, 22. Aim 2 Defining gene-gene and gene-environment interactions: Exploiting the large effect sizes and the scalability of the zebrafish, we will test gene-gene and gene-environment (lipid lowering or antihypertensive drugs, and sex hormones) interactions that are beyond the power of the available human datasets. The results of quantitative zebrafish analyses will then be directly tested in the original human data. This application will rigorously test the utility of combining GWAS data with in vivo zebrafish biology for the prioritization and functional evaluation of genes within loci. The work will allow the exploration of gene-gene and gene-drug interactions. We also anticipate that the multi-system exploration of the effects of genetic manipulations in the zebrafish will also inform our understanding of clinical phenotypes. Finally, these studies will lay the foundation for exploration of the gene networks underlying common cardiac and vascular phenotypes, and establish high-throughput biology in the zebrafish as a platform to complement GWAS. PUBLIC HEALTH RELEVANCE: With the completion of the Human Genome Project there has been a wave of genetic studies, generating large numbers of new potential disease genes. Understanding how these genes work, alone, together or in concert with the environment, to cause each disease requires new approaches which allow the study of complex effects on different tissues or organs, but offer sufficient throughput to deal with literally hundreds of genes. The zebrafish can model many human diseases, and in this application a multidisciplinary team will integrate insights from zebrafish biology and the Framingham Heart Study to explore the genetics of major cardiovascular diseases.